In-fusion joining few parts together - possible? ideas? (Oct/10/2010 )

Hi all,

I had read in http://openwetware.org/wiki/In-fusion_biobrick_assembly where the method was adding one fragment into another "fragment+vector"

For some reason, I have to join a few fragments (~500 to 900 bp) into one linearized long fragment.

Fragment A generated by 2 primers: Fwd1(homologous to vector left end), Rev1

Fragment B generated by 2 primers: Fwd2(homologous Rev1), Rev2

Fragment C generated by 2 primers: F3(homologous Rev2), Rev3(homologous to vector right end)

Can I addall 3 purified fragment (A, B, C) with one linearized vector and added the whole into one single tube of in-fusion reaction, and expect to get a complete vector like: Possible?

5'----A-B-C------C'

Or if such a case, I shall just do a fusion/joint/ assemble PCR to join 3 fragment together before adding into in-fusion reaction?


Thank you and appreciate all your ideas.

Adrian

anyone got any idea? Or should I explain further my question?

In principle, you should be able to do this all at once. I don't have a feeling for the likely efficiency of the process, however.

Hi phage434, thanks for your reply.

In other words, is that meaning to say that the efficiency of process was reduced by:
1) too much variety of fragments, or
2) the concentration of total fragments as it had been reduced, or
3) too much DNTP nucleotides generated during the "chew back" process?

Thanks again.